The present invention, in some embodiments thereof, relates to downregulation of microRNA-7 and, more particularly, but not exclusively, to the use of same for promoting insulin production from pancreatic beta cells.
The development of the endocrine pancreas is governed by a network of transcription factors that specify the different endocrine cell types, including insulin-producing beta cells, glucagon-producing alpha cells, delta cells (somatostatin producing cells), PP cells (pancreatic peptide producing cells) and epsilon cells (ghrelin producing cells). The transcription factor Neurogenin3 (Ngn3) initiates the endocrine differentiation program and then a complex network of transcription factors is activated to differentially specify the endocrine lineages.
Pax6 is one such transcription factor acting downstream of Ngn3. Pax6 is pivotal in the differentiation of pancreatic beta-cells and alpha-cells, as islet morphogenesis has been shown to be disrupted when Pax6 expression is attenuated. In both humans and mice, two Pax6 alleles are required in order to maintain glucose homeostasis and loss of one allele results in glucose intolerance. The development of multiple other organs is sensitive to Pax6 haplo-insufficiency, including the iris and the lens. Normal embryonic development cannot tolerate high levels of Pax6. Thus, for example, Pax6 overexpression in mice causes eye abnormalities and induces apoptosis in the brain and the endocrine pancreas. Thus, it appears that Pax6 expression is tightly controlled to ensure appropriate levels of expression.
Genome-encoded miRNAs bind to specific sites on the 3′ untranslated region (3′UTR) of their target mRNAs, to impart posttranscriptional silencing. This regulatory layer acts in concert with transcription factors to refine gene expression and confer robustness to developmental transitions. Total inactivation of miRNA maturation causes pancreas agenesis [Lynn et al. (2007) Diabetes 56(12): 2938-45], indicating that miRNAs are essential for early pancreas development. Melkman-Zehavi et al. disclosed that miRNAs control insulin content in pancreatic beta cells by downregulation of transcriptional repressors, thus allowing reactivation of insulin transcription [Melkman-Zehavi et al. (2011) EMBO Journal 1-11]. Furthermore, specific miRNAs were shown to control insulin synthesis and exocytosis in differentiated cells. For example, loss of function of miR-375 in mice disrupts islet morphogenesis and endocrine cell differentiation [Poy et al., (2009) Proc Natl Acad Sci USA (106) 5813-5818] while specific knockdown of miR-24, miR-26, miR-182 or miR-148 in beta cells downregulates insulin promoter activity and insulin mRNA levels [Melkman-Zehavi et al. (2011), supra].
miR-7 is another miRNA that is highly and specifically expressed in the endocrine pancreas in mice and humans [Bravo-Egana et al. (2008) Biochem Biophys Res Commun (366) 922-926; Correa-Medina et al. (2009) Gene Expr Patterns (9) 193-199]. miR-7 is an evolutionarily conserved miRNA, encoded by three different genomic loci in humans and mice (mouse: mmu-mir-7a-1 at Chr13, mmu-mir-7a-2 at Chr7 and mmu-mir-7b at Chr17). The duplication of the miR-7 gene in vertebrates hampers genetic loss-of-function analysis.
PCT Publication No. WO 2009/067644 (to Pastori Ricardo et al.) discloses that mir-7 is a marker of differentiated endocrine cells and plays a role in β-cell biogenesis. WO 2009/067644 further discloses that inhibition of mir-7 activity in the fetal pancreas results in inhibition of formation of insulin in the fetal pancreas.